A Metallo-Β-Lactamase Enzyme for Internal Detoxification of The

www.nature.com/scientificreports OPEN A metallo‑β‑lactamase enzyme for internal detoxifcation of the antibiotic thienamycin Seydina M. Diene1,3, Lucile Pinault2,3, Sophie Alexandra Baron2,3, Saïd Azza1,3, Nicholas Armstrong2,3, Linda Hadjadj1,3, Eric Chabrière1,3, Jean‑Marc Rolain1,3, Pierre Pontarotti1,3,4 & Didier Raoult1,3* Thienamycin, the frst representative of carbapenem antibiotics was discovered in the mid‑1970s from soil microorganism, Streptomyces cattleya, during the race to discover inhibitors of bacterial peptidoglycan synthesis. Chemically modifed into imipenem (N‑formimidoyl thienamycin), now one of the most clinically important antibiotics, thienamycin is encoded by a thienamycin gene cluster composed of 22 genes (thnA to thnV) from S. cattleya NRRL 8057 genome. Interestingly, the role of all thn‑genes has been experimentally demonstrated in the thienamycin biosynthesis, except thnS, despite its annotation as putative β‑lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo‑β‑lactamase fold proteins. Compared to known β‑lactamases such as OXA‑48 and NDM‑1, ThnS exhibited a lower afnity and less efciency toward penicillin G and cefotaxime, while imipenem is more actively hydrolysed. Moreover, like most MBL fold enzymes, additional enzymatic activities of ThnS were detected such as hydrolysis of ascorbic acid, single strand DNA, and ribosomal RNA. ThnS appears as a MBL enzyme with multiple activities including a specialised β‑lactamase activity toward imipenem. Thus, like toxin/antitoxin systems, the role of thnS gene within the thienamycin gene cluster appears as an antidote against the produced thienamycin. Bioactive compounds such as secondary metabolites are biosynthesised by non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS), enzymes that have been identifed in the diferent domains of life with an extraordinary diversity and more than 3,300 NRPS/PKS have been reported from 991 diferent organisms 1. Tese bioactive compounds exhibiting a wide range of biological activities can include siderophores, pigments, cytostatics, immunosuppressants, toxins, and antibiotics 2,3. Toxins or natural antibiotics are expressed by some microorganisms (e.g. fungus and/or bacteria) to master their ecosystems 4. According to the mode of action, antibiotics can be classifed into several groups including synthesis inhibitors of cell walls, proteins, DNA, RNA, and others5. Most antibiotic classes including aminoglycosides, macrolides, tetracyclines, chloramphenicol, and β-lactams have been synthesised by biosynthesis gene clusters identifed in Actinobacteria such as Nocardia, Sac- charopolyspora, Kitasatospora, or Streptomyces5. Furthermore, some β-lactams, especially carbapenem antibiotics, can be synthesised by more common bacteria such as Enterobacterial phytopathogens, including Serratia sp. or Erwinia carotovora subsp. atroseptica (now renamed Pectobacterium carotovora subsp. atroseptica), or Photorhabdus luminescens6,7. Like toxin-antitoxin systems, these microorganisms develop self-resistance mechanisms to protect themselves against attacks from their own biosynthetic bioactive compounds 5,8. Tese self- resistance mechanisms such as antibiotic efux pumps, antibiotic-modifying enzymes, or antibiotic-hydrolysing enzymes are ofen encoded by genes located within the biosynthesis gene clusters5. Among these antibiotics, thienamycin, the frst to be discovered and characterised as a carbapenem and one of the most clinically important antibiotic classes, is encoded by a thienamycin gene cluster (TGC) composed of 22 genes (thnA to thnV) from Streptomyces cattleya NRRL 8057 genome9,10. In addition to thienamycin, another antibiotic. cephamycin (cephalosporin sub-class) encoded by a cephamycin gene cluster (CGC) (composed of 16 genes), has been also identifed within the same S. cattleya NRRL 8057 genome 10. However, while the role of almost all genes of the TGC have been experimentally well characterised in thienamycin biosynthesis 11,12, the role and function of the 1IRD, APHM, MEPHI, IHU-Méditerranée Infection, Aix Marseille Univ, 19-21 Boulevard Jean Moulin, 13005 Marseille, France. 2Publique-Hôpitaux de Marseille (AP-HM), IHU-Méditerranée Infection, Marseille, France. 3IHU-Méditerranée Infection, Marseille, France. 4CNRS, Marseille, France. *email: didier.raoult@ gmail.com Scientifc Reports | (2021) 11:10062 | https://doi.org/10.1038/s41598-021-89600-x 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Genetic environment of the thienamycin gene cluster from the megaplasmid of Streptomyces cattleya NRRL8057. thnS gene, despite its annotation as putative β-lactamase, remain unreported and it was suggested this should be clarifed12. Here, we investigate the phylogenetic relationship of the TnS enzyme with bacterial β-lactamase enzymes and explore its enzymatic activity against diferent substrates including antibiotic drugs, ascorbic acid, DNA, and RNA substrates. Results Origins of ThnS enzyme. Te thnS gene is part of the thienamycin gene cluster (TGC) located on the linear megaplasmid (pSCAT, 1′809′491-bp, 73.21%CG) of the S. cattleya NRRL 8057 genome. As shown in Fig. 1, the TGC, with size of 28.25-Kb and %GC content of 73.61%, is composed of 22 genes in which thnI and thnU encode for the transcriptional activators of expression of the thnH, J, K, L, M, N, O, P, and Q genes, while the expression of the remaining genes (thnA-G, thnR-V) are thnIU-independent12. BlastP analysis of the TnS sequence against the NCBI database reveals the best homologous sequences (coverage ≥ 50% and similarity ≥ 84%) with metallo-β-lactamase (MBL), fold metallo-hydrolase and the L-ascorbate metabolism protein UlaG, all identifed from the Pseudonocardiaceae family. Its best homologous sequences within more common bacteria, especially Enterobacteriaceae species, are also all annotated as MBL fold metallo-hydrolase with homologies ranging from 27% coverage and 34% identity with Serratia fcaria and 79% coverage and 24.15% identity with Escherichia coli (Suppl. Table S1). Phylogenetic relationship of the ThnS enzyme with bacterial β‑lactamases. Te inferred phylogenetic tree of the TnS sequence with the four bacterial classes of β-lactamases (A, B, C, and D) shows that the TnS enzyme is closely related to the bacterial class B β-lactamases (Fig. 2A). Tis membership is confrmed by sequence alignment with the three sub-classes (B1, B2, B3) highlighting the conserved “HxHxDH” motif and H196, H263 residues, that are specifc to this metallo-β-lactamase (MBL) superfamily group (Fig. 2B). Moreover, three-dimensional (3D) structural analysis of the TnS protein model from the Phyre2 investigator database confrms with 100% confdence and more than 81% coverage that TnS was similar to the crystal structure of an uncharacterised metallo protein from Vibrio cholerae with β-lactamase-like fold (Phyre2 ID: c3bv6D; Uniprot ID: Q9KMS2). β‑lactam‑hydrolysing activity. To evaluate the enzymatic activity of TnS, this latter and two bacterial β-lactamases including class D OXA-48 and class B NDM-1 carbapenemases were expressed and tested Scientifc Reports | (2021) 11:10062 | https://doi.org/10.1038/s41598-021-89600-x 2 Vol:.(1234567890) www.nature.com/scientificreports/ Figure 2. Phylogenetic relationship of TnS with bacterial β-lactamases. (A) phylogenetic tree analysis of the TnS protein with described bacterial β-lactamase sequences; (B) protein alignment of TnS with class B β-lactamase sequences exhibiting the conserved motif and residues of this class. Te phylogenetic tree was inferred using the maximum-likelihood method in FastTree then displayed using the FigTree sofware v1.4.4 (http:// tree. bio. ed. ac. uk/ sofw are/ fgtr ee/). against diferent antibiotic substrates including nitrocefn, penicillin G, cefotaxime, and imipenem. Interestingly, TnS was unable to hydrolyze the nitrocefn substrate while both bacterial β-lactamases (OXA-48 and NDM-1) signifcantly hydrolyse this substrate (data not shown). However, as presented in Fig. 3A,B, the TnS enzyme degrades penicillin G, as observed for OXA-48 and NDM-1 enzymes, but more slowly during the 24 h test, sug- gesting a lower afnity toward penicillin G. Regarding imipenem hydrolysis, the TnS activity was comparable with both OXA-48 and NDM-1 carbapenemases (Fig. 3C). TnS here appears to be more active against this drug since the measured amount of imipenem metabolite (imipenemoic acid) afer 24 h of incubation was higher for TnS compared to the two enzyme controls (Fig. 3D). Compared to both bacterial β-lactamases, the hydrolysis of cefotaxime by TnS was detected only afer 24 h and appears then less active against this cephalosporin compared to the enzyme controls, also suggestive of a lower afnity toward cephalosporin antibiotic (Fig. 3E). Degradation activity on ascorbic acid. Based on BlastP analyses (presented above), the TnS enzyme exhibited signifcant homology with UlaG, a manganese-dependent metallo-β-lactamase enzyme involved in L-ascorbate metabolism through the degradation of ascorbic acid13. Interestingly, it has been suggested that the UlaG precursor shared the RNA metabolising function with the last common ancestor enzyme and its descend- ants have a wide functional activities and phylogenetic distribution13. Figure 4A, TnS enzyme shows 3D structure similarity with UlaG protein from E. coli and the same amino acid residues in their catalytic site

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